Aniridia Patients Research Articles

Genetic analysis using next-generation sequencing and multiplex ligation probe amplification in Chinese aniridia patients

BackgroundCongenital aniridia is a rare pan-ocular disease characterized by complete irideremia, partial iridocoloboma. The progressive nature of aniridia is frequently accompanied by secondary ocular complications such as glaucoma and aniridia-associated keratopathy, which can lead to severely impaired vision or blindness. The genetic basis of aniridia has been the subject of numerous studies, leading to the development of innovative therapeutic options based on PAX6 nonsense mutations. Specific knowledge of the genetics of aniridia has become increasingly important. To report the clinical features, elucidate the genetic etiology, and reveal the mutational spectrum of congenital aniridia in the Chinese population, sixty patients with congenital aniridia from 51 families were recruited. Candidate genes associated with developmental eye diseases were identified and analyzed using panel-based next-generation sequencing (NGS), and mutations were confirmed through polymerase chain reaction and Sanger sequencing. Multiplex ligation probe amplification (MLPA) of PAX6 and FOXC1 was performed to detect copy number variations in the patients without intragenic mutations.ResultsClinical examination revealed complete iris hypoplasia in 58 patients and partial iris hypoplasia in two patients. Additionally, two patients were diagnosed with Wilms’ tumor-aniridia-genital anomalies-retardation syndrome and nephroblastoma. By combining panel-based NGS and MLPA, 43 intragenic mutations or deletions of PAX6, FOXC1, and BCOR were identified in 59 patients, including 33 point mutations (76.7%) in 43 patients and 10 deletions (23.3%) in 16 patients. The total detection rate was 98.3%. Phenotypic variation was observed between and within families.ConclusionsVariations in PAX6 and its adjacent regions were the predominant causes of aniridia in China. In addition to intragenic point mutations in PAX6, deletion of PAX6 or its adjacent genes is a common cause of congenital aniridia. Furthermore, FOXC1 is an important gene associated with congenital aniridia. The combination of panel-based NGS and MLPA significantly enhanced the detection rate of gene mutations in patients with congenital aniridia.

838 Review of the Literature on Iris Implantation in Aniridia Patients: Techniques and Clinical Outcomes

838 Review of the Literature on Iris Implantation in Aniridia Patients: Techniques and Clinical Outcomes

Optic Disc Hypoplasia Assessment in PAX6 -Related Aniridia.

This study aims to characterize optic disc hypoplasia in congenital aniridia using ultra-wide-field imaging (UWFI) and nonmydriatic retinal photography (NMRP). We also investigated the relation between optic disc hypoplasia and foveal hypoplasia. This is a retrospective case series of patients diagnosed with PAX6 -related aniridia in a National Referral Center, who underwent UWFI, NMRP, and spectral-domain optical coherence tomography (SD-OCT) . The disc diameter (DD) and the disc-to-fovea distance (DF) were measured. The DD:DF ratio was used to assess the relative size of the optic disc. The analyses were carried with respect to paired age- and sex-matched healthy controls. SD-OCT was used for foveal hypoplasia grading (from 1 to 4) and retinal nerve fiber layer (RNFL) analysis. Mean manual DD:DF ratio was 0.33 (95% CI: 0.31-0.35) in aniridia patients versus 0.37 (95% CI: 0.36-0.39) in control patients (n = 20, P = 0.005) measured on NMRP and 0.32 (95% CI: 0.30-0.35) in aniridia patients versus 0.37 (95% CI: 0.37-0.39) in control patients (n = 26, P < 0.0001) when assessed on UWFI. Mean semiautomated DD:DF ratio measured on UWFI in aniridia patients was 0.31 (95% CI: 0.29-0.33) versus 0.37 (95% CI: 0.36-0.38) in control patients ( P < 0.0001). Also, a negative correlation was found significant between the grade of foveal hypoplasia and the mean semiautomated DD:DF ratio (r = -0.52, 95% CI: -0.76 to -0.15, P = 0.0067). Finally, a significant negative correlation was found between the peripapillary temporal RNFL thickness and the grade of foveal hypoplasia ( P = 0.0034). The DD:DF ratio is significantly reduced in PAX6 -related aniridia patients and correlates with the severity of foveal hypoplasia. This ratio is a valuable tool for optic disc hypoplasia assessment in congenital aniridia, especially when provided semiautomatically by UWFI.

Altered cell fates of limbal stem cells in aniridia associated with PAX6 mutations

Proper biological function of stem cells in tissues and organs relies on their correct cell fates, and transcription factors (TFs) play key roles in controlling cell fate. Altered cell fates of stem cells due to genetic mutations or other factors can lead to developmental and homeostatic abnormalities. Aniridia is caused by mutations that affect the transcription factor PAX6, and patients with aniridia suffer from congenital and progressive corneal opacification. This is at least partly due to deficient function of limbal stem cells (LSCs) located in the periphery of the cornea. To test the hypothesis that corneal opacity is associated with altered cell fates in LSCs of aniridia patients, we performed transcriptome analyses using two different types of stem cells derived from aniridia patients: induced pluripotent stem cells (iPSCs) and primary limbal stem cells (pLSCs). First, we differentiated iPSCs to obtain induced LSCs. Our transcriptome data showed that induced LSCs derived from aniridia patients do not have the same epithelial identity as normal cells, but instead have an enhanced neural or pluripotent identity. In parallel, we investigated whether patient‐derived pLSCs could adopt the cell fate of a similar epithelial cell type, specifically skin keratinocytes, which give rise to the non‐transparent epidermis. By comparing the transcriptomes of these two cell types, we observed a clear loss of the normal LSC fate in the aniridia pLSCs as compared to normal pLSCs. However, the gain of keratinocyte cell fate was not evident. These findings demonstrate the essential role of PAX6 function in determining the correct cell fate of LSCs.

Identification of duloxetine to treat aniridia‐related keratopathy: Link to inflammation?

Haploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by iris hypoplasia and reduced visual acuity. Patients have also progressive disorders including aniridia‐related keratopathy (ARK), caused by a combination of factors including limbal stem‐cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub‐epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. There is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation. We used genome editing (CRISPR/Cas9) to introduce a nonsense mutation into one allele of the PAX6 gene in limbal stem cells.The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also altered. Of interest, recombinant PAX6 protein was sufficient to rescue the phenotype. By High Throughput Screening of a FDA‐approved library, we identified Duloxetine, a selective serotonin and norepinephrine reuptake inhibitor antidepressant, that rescued both PAX6 endogenous protein level and altered cell migration of the mutant limbal stem cells, through specific inhibition of the ERK/MEK pathway. Furthermore, we found that Duloxetine modulates inflammation through activation of Pax6, strongly suggesting that Pax6 could be a modulator of neuroinflammation. Since Duloxetine is already used as anti‐psychotropic drugs, its repurposed use to treat topically aniridia eye and other ocular inflammatory diseases could become realistic.

Identification of duloxetine to treat aniridia‐related keratopathy: Link to inflammation?

Haploinsufficiency of PAX6 in humans is the main cause of congenital aniridia, a rare eye disease characterized by iris hypoplasia and reduced visual acuity. Patients have also progressive disorders including aniridia‐related keratopathy (ARK), caused by a combination of factors including limbal stem‐cell deficiency, impaired healing response, abnormal differentiation, and infiltration of conjunctival cells onto the corneal surface. It usually begins in the first decade of life resulting in recurrent corneal erosions, sub‐epithelial fibrosis with corneal decompensation and opacification. Unfortunately, current treatment options for aniridia patients are currently limited. There is no in vitro cellular model of AKT needed for drug/therapeutic tools screening and validation. We used genome editing (CRISPR/Cas9) to introduce a nonsense mutation into one allele of the PAX6 gene in limbal stem cells.The mutated cells displayed reduced cell proliferation and cell migration but enhanced cell adhesion. Known PAX6 targets expression was also altered. Of interest, recombinant PAX6 protein was sufficient to rescue the phenotype. By High Throughput Screening of a FDA‐approved library, we identified Duloxetine, a selective serotonin and norepinephrine reuptake inhibitor antidepressant, that rescued both PAX6 endogenous protein level and altered cell migration of the mutant limbal stem cells, through specific inhibition of the ERK/MEK pathway. Furthermore, we found that Duloxetine modulates inflammation through activation of Pax6, strongly suggesting that Pax6 could be a modulator of neuroinflammation. Since Duloxetine is already used as anti‐psychotropic drugs, its repurposed use to treat topically aniridia eye and other ocular inflammatory diseases could become realistic.

Secondary Data Analysis of Inflammation-Related mRNAs in Conjunctival Impression Cytology Samples of Aniridia Patients.

Aniridia is a rare corneal disease that is often associated with aniridia-associated keratopathy (AAK). In AAK, the conjunctival tissue crosses the limbal border, forming a corneal pannus that extends into the corneal center. With increasing AAK severity, corneal pannus formation, vascularization, and ocular surface inflammation increase. The purpose of this study was to investigate inflammation-related mRNA expression in conjunctival epithelial cells in AAK and its relationship with AAK severity. Using impression cytology, bulbar conjunctival cells were sampled from 20 subjects with congenital aniridia and 20 age-matched and sex-matched healthy control subjects. RNA was extracted, and mRNA analyses were performed using microarray, which was evaluated for inflammatory markers. In the analyzed aniridia subjects, 70 deregulated mRNAs encoding proinflammatory or antiinflammatory cytokines or factors associated with chronic inflammation, including increased IL-1, IL-8, and MIP3A/CCL20 mRNA. The most downregulated mRNA was TIMP3, and the most upregulated mRNA was Protein c-Fos.Of the 70 mRNAs, 14 inflammation-related genes were altered only in the mild AAK forms, whereas only 2 mRNAs were altered only in the severe AAK forms (TLR4 and PPARG). The expression of numerous proinflammatory and antiinflammatory cytokines is deregulated at the ocular surface of aniridia subjects with mild AAK. Thus, early antiinflammatory treatment may prevent or slow down corneal scarring and pannus formation in aniridia subjects.

Characterization of neural damage and neuroinflammation in Pax6 small-eye mice

Aniridia is a panocular condition characterized by a partial or complete loss of the iris. It manifests various developmental deficits in both the anterior and posterior segments of the eye, leading to a progressive vision loss. The homeobox gene PAX6 plays an important role in ocular development and mutations of PAX6 have been the main causative factors for aniridia. In this study, we assessed how Pax6-haploinsufficiency affects retinal morphology and vision of Pax6Sey mice using in vivo and ex vivo metrics. We used mice of C57BL/6 and 129S1/Svlmj genetic backgrounds to examine the variable severity of symptoms as reflected in human aniridia patients. Elevated intraocular pressure (IOP) was observed in Pax6Sey mice starting from post-natal day 20 (P20). Correspondingly, visual acuity showed a steady age-dependent decline in Pax6Sey mice, though these phenotypes were less severe in the 129S1/Svlmj mice. Local retinal damage with layer disorganization was assessed at P30 and P80 in the Pax6Sey mice. Interestingly, we also observed a greater number of activated Iba1+ microglia and GFAP + astrocytes in the Pax6Sey mice than in littermate controls, suggesting a possible neuroinflammatory response to Pax6 deficiencies.

Long-Term Clinical Outcomes of Ahmed Valve Implantation in Aniridic Glaucoma.

This study assessed the efficacy and safety of Ahmed valve implantation in patients with aniridic glaucoma for three consecutive years. Six adult patients (seven eyes) with Ahmed valve (AV) implants for aniridic glaucoma were enrolled in the study. The primary outcome measures were intraocular pressure reduction, glaucoma medication use, success rates, and visual acuity after AV implantation. A 30% reduction in IOP from baseline without the need for re-intervention was considered an effective treatment. The cessation of antiglaucoma medications was defined as complete success. Intraoperative and postoperative complications were included as secondary outcome measures. Measurements were performed preoperatively, at the first week, and 1, 3, 6, 12, 18, 24, 30, and 36 months postoperatively. A total of seven eyes (6 patients) were evaluated 36 months after AV implantation. The mean ± SD values of IOP preoperatively at 1 day, 1 week, and 1, 3, 6, 12, 18, 24, 30, and 36 months postoperatively were 30.4 ± 4.0 mmHg, 14.6 ± 4.6 mmHg, 16.1 ± 4.6 mmHg, 20.7 ± 7.0 mmHg, 14.5 ± 2.7 mmHg, 16.5 ± 5.9 mmHg, 16.2 ± 4.0 mmHg, 16.3 ± 4.3 mmHg, 17.2 ± 10.1 mmHg, 17.6 ± 6.9 mmHg, and 18.2 ± 5.5 mmHg, respectively. At the last follow up, the mean IOP was reduced by 40.2%. The qualified success rate was 85.7%. One patient (one eye) at the last follow-up visit did not require antiglaucoma medications, resulting in a complete success rate of 14.3%. Intra- and postoperative mild or moderate subconjunctival bleeding was observed in all the patients. No other major/minor intraoperative or postoperative complications were noted. In long-term follow up, the AV implantation procedure is well-tolerated and relatively safe for reducing IOP in adult aniridia patients with glaucoma. These results should be validated through studies involving a larger patient cohort.

Minimally invasive approach for the management of tube–corneal touch in aniridic glaucoma

Aniridic glaucoma, with a prevalence of around 50% in aniridia patients, often requires surgical management with glaucoma drainage device implantation. It is associated with many complications related to the tube or plate. We present a case of tube endothelial touch in aniridic glaucoma where repositioning of the Ahmed glaucoma valve tube was performed using transcameral prolene suture fixation. The advantage of this technique is that it is minimally invasive and maintains the stability of the anterior chamber throughout the surgery.

Restoration of functional PAX6 in aniridia patient iPSC-derived ocular tissue models using repurposed nonsense suppression drugs

Congenital aniridia is a rare, pan-ocular disease causing severe sight loss, with only symptomatic intervention offered to patients. Approximately 40% of aniridia patients present with heterozygous nonsense variants in PAX6, resulting in haploinsufficiency. Translational readthrough-inducing drugs (TRIDs) have the ability to weaken the recognition of in-frame premature termination codons (PTCs), permitting full-length protein to be translated. We established induced pluripotent stem cell (iPSC)-derived 3D optic cups and 2D limbal epithelial stem cell (LESC) models from two aniridia patients with prevalent PAX6 nonsense mutations. Both invitro models show reduced PAX6 protein levels, mimicking the disease. The repurposed TRIDs amlexanox and 2,6-diaminopurine (DAP) and the positive control compounds ataluren and G418 were tested for their efficiency. Amlexanox was identified as the most promising TRID, increasing full-length PAX6 levels in both models and rescuing the disease phenotype through normalization of VSX2 and cell proliferation in the optic cups and reduction of ABCG2 protein and SOX10 expression in LESCs. This study highlights the significance of patient iPSC-derived cells as a new model system for aniridia and proposes amlexanox as a new putative treatment for nonsense-mediated aniridia.

Optical Coherence Tomography Angiography Assessment in Congenital Aniridia

This study aims to characterize foveal vasculature assessed by optical coherence tomography angiography (OCT-A) in congenital aniridia which is hallmarked by foveal hypoplasia (FH). Cross-sectional case-control analysis. At the National Referral Center for congenital aniridia, patients with confirmed PAX6-related aniridia and FH diagnosed on spectral-domain OCT (SD-OCT) with available OCT-A and matched control subjects were included. OCT-A was performed in patients with aniridia and control subjects. Foveal avascular zone (FAZ) and vessel density (VD) were collected. VD in the foveal and parafoveal areas at the level of the superficial and deep capillary plexi (SCP and DCP, respectively) were compared between the 2 groups. In patients with congenital aniridia, correlation between VD and the grading of FH was assessed. Among 230 patients with confirmed PAX6-related aniridia, high-quality macular B-scans and OCT-A were available in 10 patients. On the foveal area, mean VD was higher in aniridia patients (41.10%, n=10) than in control subjects (22.65%, n=10) at the level of the SCP and the DCP (P=.0020 and P=.0273, respectively). On the parafoveal area, mean VD was lower in patients with aniridia (42.34%, n=10) than in healthy subjects (49.24%, n=10) at the level of both plexi (P=.0098 and P=.0371, respectively). In patients with congenital aniridia, a positive correlation was found between the grading of FH and the foveal VD at the SCP (r=0.77, P=.0106). Vasculature is altered in PAX6-related congenital aniridia, higher in foveal and lower in parafoveal areas, especially when FH is severe, which is consistent with the concept that the absence of retinal blood vessels is essential for foveal pit development.

A new association of PAX6 variation with Juvenile onset open angle glaucoma.

Mutations in the PAX6 gene are generally associated with aniridia. We describe a family with Juvenile onset open angle glaucoma (JOAG), where one of the two children had JOAG and the other Juvenile ocular hypertension. Whole exome sequencing was performed for the parents and their two affected children where the proband and her sibling were detected to have a de novo PAX6 gene variant in the absence of aniridia. All previously described gene mutations for glaucoma were looked for in the family. The potential pathogenicity of the identified variants was assessed by determining their frequency in large public exome databases; as well as using the current ACMG guidelines. The same heterozygous variant at NM_000280.6:c.1124 C > A; p. Pro375Gln in the PAX6 gene was detected in the proband and her affected brother. The variant has been described in aniridia patients before and has been shown to cause a weaker DNA binding using functional studies. This report expands the phenotypic spectrum of the PAX6 gene to include Juvenile onset open angle glaucoma.

PAX6 disease models for aniridia.

Aniridia is a pan-ocular genetic developmental eye disorder characterized by complete or partial iris and foveal hypoplasia, for which there is no treatment currently. Progressive sight loss can arise from cataracts, glaucoma, and aniridia-related keratopathy, which can be managed conservatively or through surgical intervention. The vast majority of patients harbor heterozygous mutations involving the PAX6 gene, which is considered the master transcription factor of early eye development. Over the past decades, several disease models have been investigated to gain a better understanding of the molecular pathophysiology, including several mouse and zebrafish strains and, more recently, human-induced pluripotent stem cells (hiPSCs) derived from aniridia patients. The latter provides a more faithful cellular system to study early human eye development. This review outlines the main aniridia-related animal and cellular models used to study aniridia and highlights the key discoveries that are bringing us closer to a therapy for patients.

Congenital Aniridia and Ocular motility

To study the frequency and types of strabismus in congenital aniridia, the presence of associated nystagmus, foveal hypoplasia, and congenital cataracts. Prospective, single-center cohort study. A review was conducted of 379 medical records of congenital aniridia patients who had a follow-up at the Necker-Enfants malades University Hospital between 2006 and 2022; the target age was between 12 months and 30 years. Ophthalmologic and orthoptic assessments according to age were performed. Strabismus was further analyzed according to the type of aniridia, foveal hypoplasia, and laterality of congenital cataract. Strabismus was diagnosed in 150 patients; 73 were included in the study (28 males [38%] and 45 females [62%]), with a mean age of 11.02 years. The mean follow-up was 24 months. Thirty-six (49.3%) presented with familial aniridia, and 37 (50.7%) presented with sporadic aniridia. Thirty-six (49.3%) were diagnosed with esotropia, 37 (50.7%) had exotropia. Nystagmus was detected in 70 patients (96%). Thirty-nine (53.4%) suffered from congenital cataract, 10 unilateral (25.7%) and 29 bilateral (74.3%). Foveal hypoplasia was found in 73 cases (100%); esotropia was predominant in all grades. PAX6 mutation was found in 56 patients (77%). Strabismus is one of the clinical signs of congenital aniridia. The laterality of congenital cataracts seems to affect the type of strabismus. The grade of foveal hypoplasia has little impact on strabismus but is prevalent for nystagmus. Foveal hypoplasia affects optical focus, which is essential for binocularity; this could explain the poor binocular adjustment leading to strabismus without exotropia or esotropia predominance.

Relative Frequencies of PAX6 Mutational Events in a Russian Cohort of Aniridia Patients in Comparison with the World's Population and the Human Genome.

Genome-wide sequencing metadata allows researchers to infer bias in the relative frequencies of mutational events and to predict putative mutagenic models. In addition, much less data could be useful in the evaluation of the mutational frequency spectrum and the prevalent local mutagenic process. Here we analyzed the PAX6 gene locus for mutational spectra obtained in our own and previous studies and compared them with data on other genes as well as the whole human genome. MLPA and Sanger sequencing were used for mutation searching in a cohort of 199 index patients from Russia with aniridia and aniridia-related phenotypes. The relative frequencies of different categories of PAX6 mutations were consistent with those previously reported by other researchers. The ratio between substitutions, small indels, and chromosome deletions in the 11p13 locus was within the interval previously published for 20 disease associated genomic loci, but corresponded to a higher end due to very high frequencies of small indels and chromosome deletions. The ratio between substitutions, small indels, and chromosome deletions for disease associated genes, including the PAX6 gene as well as the share of PAX6 missense mutations, differed considerably from those typical for the whole genome.

Human Oral Mucosal Fibroblasts from Limbal Stem Cell Deficient Patients as an Autologous Feeder Layer for Epithelial Cell Culture

Purpose To investigate if human oral mucosal fibroblasts (HOMF) from patients with limbal stem cell deficiency (LSCD) can be used as an autologous feeder layer to support the culture of epithelial cells for potential clinical use. Methods HOMF were isolated from oral mucosal biopsies obtained from the following groups of patients with LSCD: aniridia, mucous membrane pemphigoid (MMP), Stevens–Johnson syndrome (SJS), and ectodermal dysplasia (ED). The ability of these cells to support the culture of human limbal epithelial cells (HLE) was compared to that of HOMF from non-LSCD donors and 3T3s commonly used to culture epithelial cells for use in the clinic to treat LSCD. Results HOMF were successfully obtained by explant culture for 3/3 aniridia patients, 3/3 MMP patients, 1/3 SJS patients, and 1/1 ED patients. All HOMF cultured from these LSCD groups supported the expansion of HLE with epithelial culture times and total colony forming efficiency (CFE) comparable to those achieved on HOMF isolated from donors without LSCD. PCR showed that all HLE cultured on LSCD donor HOMF expressed p63α, CK15, PAX6, CK12, and MUC16 as did HLE cultured on the control non-LSCD donor HOMF and 3T3s. Western blotting detected CK15 and MUC16 protein expression in all groups. Conclusions HOMF from patients with LSCD can be successfully used to support the expansion of epithelial cells. These cells may therefore be useful as autologous feeder fibroblasts for the expansion of epithelial cells for use in the clinic to treat LSCD.

Long-term retinal protection by MEK inhibition in Pax6 haploinsufficiency mice

Aniridia is a panocular condition characterized by impaired eye development and vision, which is mainly due to the haploinsufficiency of the paired-box-6 (PAX6) gene. Like what is seen in aniridia patients, Pax6-deficient mice Pax6Sey-Neu/+ exhibit a varied degree of ocular damage and impaired vision. Our previous studies showed that these phenotypes were partially rescued by PD0325901, a mitogen-activated protein kinase kinase (MEK or MAP2K) inhibitor. In this study, we assessed the long-term efficacy of PD0325901 treatment in retinal health and visual behavior. At about one year after the postnatal treatment with PD0325901, Pax6Sey-Neu/+ mice showed robust improvements in retina size and visual acuity, and the elevated intraocular pressure (IOP) was also alleviated, compared to age-matched mice treated with vehicles only. Moreover, the Pax6Sey-Neu/+ eyes showed disorganized retinal ganglion cell (RGC) axon bundles and retinal layers, which we termed as hotspots. We found that the PD treatment reduced the number and size of hotspots in the Pax6Sey-Neu/+ retinas. Taken together, our results suggest that PD0325901 may serve as an efficacious intervention in protecting retina and visual function in aniridia-afflicted subjects.

Congenital aniridia and strabismus

AbstractPurposeCongenital aniridia is a panocular disease with congenital absence of iris. Congenital Aniridia affects, beyond iris, cornea, angle structures, lens and fovea, and possibly associated with other anomalies. This genetic rare disease can cause severe visual impairment occurring from various mechanisms as glaucoma, limbal insufficiency and foveal hypoplasia. PAX6 gene is mainly involved in the disease. We aim to understand the features of patients with congenital aniridia and strabismus from 1 yo to 30 yo, identifying the characteristics of oculomotor anomalies.MethodsWe analyse our 330 aniridia patients cohort in a retrospective study to identify patient with a good fixation and minimal cooperation. All patients underwent history of the disease, a complete ocular examination with ophthalmic exploration, orthoptic and had genetic molecular analysis.ResultsOf the 330 subjects cohort, 36 patients, 23 females and 13 males (range 1 yo to 30 yo) were identified and diagnosed with strabismus with mean age of 11.02 yo. A family history was reported in 55% of the patients. Marked nystagmus was present in 69% of the cases. Foveal hypoplasia from Thomas classification, was observed in all patients and Grade IV in 86% of cases. Esotropia was observed in 38.8%, exotropia in 36.1% and hypertropia in 5.5%. Associated congenital cataract is described and worsened the strabismus.ConclusionsOur study demonstrated a high frequency of strabismus in congenital aniridia. Marked nystagmus and associated congenital cataract worsened the strabismus treatment. In this rare disease, further studies are required for the follow‐up of aniridia patients in order to get better therapeutic options.

Decreased FABP5 and DSG1 protein expression following PAX6 knockdown of differentiated human limbal epithelial cells

PAX6 haploinsufficiency related aniridia is characterized by disorder of limbal epithelial cells (LECs) and aniridia related keratopathy. In the limbal epithelial cells of aniridia patients, deregulated retinoic acid (RA) signaling components were identified. We aimed to visualize differentiation marker and RA signaling component expression in LECs, combining a differentiation triggering growth condition with a small interfering RNA (siRNA) based aniridia cell model (PAX6 knock down).Primary LECs were isolated from corneoscleral rims of healthy donors and cultured in serum free low Ca2+ medium (KSFM) and in KSFM supplemented with 0.9 mmol/L Ca2+. In addition, LECs were treated with siRNA against PAX6. DSG1, PAX6, KRT12, KRT 3, ADH7, RDH10, ALDH1A1, ALDH3A1, STRA6, CYP1B1, RBP1, CRABP2, FABP5, PPARG, VEGFA and ELOVL7 expression was determined using qPCR and western blot.DSG1, FABP5, ADH7, ALDH1A1, RBP1, CRABP2 and PAX6 mRNA and FABP5 protein expression increased (p ≤ 0.03), PPARG, CYP1B1 mRNA expression decreased (p ≤ 0.0003) and DSG1 protein expression was only visible after Ca2+ supplementation. After PAX6 knock down and Ca2+ supplementation, ADH7 and ALDH1A1 mRNA and DSG1 and FABP5 protein expression decreased (p ≤ 0.04), compared to Ca2+ supplementation alone.Using our cell model, with Ca2+ supplementation and PAX6 knockdown with siRNA treatment against PAX6, we provide evidence that haploinsufficiency of the master regulatory gene PAX6 contributes to differentiation defect in the corneal epithelium through alterations of RA signalling. Upon PAX6 knockdown, DSG1 differentiation marker and FABP5 RA signaling component mRNA expression decreases. A similar effect becomes apparent at protein level though differentiation triggering Ca2+ supplementation in the siRNA-based aniridia cell model. Expression data from this cell model and from our siRNA aniridia cell model strongly indicate that FABP5 expression is PAX6 dependent. These new findings may lead to a better understanding of differentiation processes in LECs and are able to explain the insufficient cell function in AAK.